Our application of emerging tools in cryo-EM to the study of membrane proteins and their conformational changes continues to grow. Projects of current interest include studies of ionotropic glutamate receptors, prokaryotic and eukaryotic ion channels, G protein-coupled receptors, growth factor receptors and multi-drug ABC transporters. Progress on some of these projects is highlighted below. Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. The ionotropic glutamate receptors undergo a gating cycle whereby a receptor in the closed resting state is bound by glutamate at the ligand binding domain (LBD). This initiates a transition to the open state, where ions can pass through the channel. The channel then transitions to the desensitized state, where the channel remains closed, but glutamate is not released from the LBD. Previously, we showed that when AMPA receptors transition from the resting to the active open state, there is a corkscrew motion of the receptor assembly, driven by closure of the ligand binding domain. Where the existing crystal structure of the AMPA receptor GluA2, which had a significantly shortened linker between the amino terminal domain (ATD) and the membrane-proximal ligand binding domain (LBD), indicated there was a large buried interface between the ATD and LBD, our structure of the full-length GluA2 showed a significant gap between those domains, which allows for this large scale movement. We also showed that transition from the open to desensitized state is accompanied by rupture of the amino terminal domain tetramer in AMPA, but not kainate receptors, with a 2-fold to 4-fold symmetry transition in the ligand binding domains in both subtypes. Our 7.6 Angstrom structure of a desensitized kainate receptor GluK2 showed how these changes accommodate channel closing. Following up on this initial work, this past year we determined the structure of the kainate receptor GluK2 at 3.8 Angstrom resolution in its desensitized state, which was sufficient to build a de novo model of the protein. Our high-resolution structure revealed a desensitization ring, a series of helices between the LBDs, which surround the top of the central pore of the channel. These helices coordinate the maintenance of the desensitized state, and preserved the closed conformation of the LBDs; our study suggests that release of ligand from the LDB initiates a breaking of the desensitization ring, which allows the channel to revert to the resting state. We are also making progress on structural studies of human P-glycoprotein, a small, pseudosymmetric member of the ABC transporter family that is responsible for the export of a variety of small molecule compounds. While we were unable to achieve high resolution with our structures of this very small, highly flexible integral membrane protein, our analysis did reveal a conformational landscape for the ATPase cycle of this protein. We found that, unexpectedly, the protein samples both the open (nucleotide binding domains separated) and closed (nucleotide binding domains close together) conformations in the apo and ATP-bound states. In the post-hydrolysis state, we found that the protein is locked in a single, closed conformation; after release of phosphate, and before exchange of ADP for ATP, P-glycoprotein samples a continuum of open conformations. While we continue efforts to obtain higher resolution, the closed conformation discovered in our present work already represent a novel structure that does not match any existing crystal structures for a member of the ABC transporter family. While the work on G protein coupled receptors and growth factor receptors is still at an early phase, we are actively pursuing the development of general methods to study membrane protein structures by cryo-EM. While the most common method for solubilization is detergent (such as DDM, which was used for the high resolution structures of GluK2 and the CorA ion channel), we are also actively exploring the use of amphipols and nanodiscs, which potentially offer a more native-like membrane environment.